Characterization of a Direct Oxygen Sensor Heme Protein fromEscherichia coli

Abstract

A protein containing a heme-binding PAS (PAS is from the protein names in which imperfect repeat sequences were first recognized: PER, ARNT, and SIM) domain from Escherichia coli has been implied a direct oxygen sensor (Ec DOS) enzyme. In the present study, we isolated cDNA for the Ec DOS full-length protein, expressed it in E. coli, and examined its structure-function relationships for the first time. Ec DOS was found to be tetrameric and was obtained as a 6-coordinate low spin ferric heme complex. Its ␣-helix content was calculated as 53% by CD spectroscopy. The redox potential of the heme was found to be ؉67 mV versus SHE. Mutation of His-77 of the isolated PAS domain abolished heme binding, whereas mutation of His-83 did not, suggesting that His-77 is one of the heme axial ligands. Ferrous, but not ferric, Ec DOS had phosphodiesterase (PDE) activity of nearly 0.15 min ؊1 with cAMP, which was optimal at pH 8.5 in the presence of Mg 2؉ and was strongly inhibited by CO, NO, and etazolate, a selective cAMP PDE inhibitor. Absorption spectral changes indicated tight CO and NO bindings to the ferrous heme. Therefore, the present study unequivocally indicates for the first time that Ec DOS exhibits PDE activity with cAMP and that this is regulated by the heme redox state.Heme proteins and enzymes perform a broad range of functions. Well known examples include O 2 storage with myoglobin, O 2 carriage with hemoglobin, mediators of electron transfer with cytochromes, and catalytic activation of heme ligands with P450s and peroxidases (1-3). Recently, a new class of heme enzymes involved in intramolecular signal transduction is emerging, known as heme-based sensors (4 -6). Almost of all the heme-based sensors contain two different functional domains as follows: one is an N-terminal heme domain, which acts a sensor, and the other is a catalytic domain such as a histidine kinase or a soluble guanylate cyclase. These heme sensor enzymes use the heme for mediating transcriptional and regulatory events associated with the presence of gaseous molecules such as CO, NO, and O 2 (4 -6). In these enzymes, the ligand association or dissociation from the heme iron leads to protein conformational changes, which transmit signals to the other domain where they initiate catalytic function or DNA binding. For example, the CooA 1 protein from Rhodospirillum rubrum is a CO sensor heme protein that regulates the expression of the coo genes associated with CO-dependent growth (Refs. 7 and 8 and references therein). Soluble guanylate cyclase is an NO sensor heme protein that regulates conversion of 5Ј-GTP to the intracellular second messenger, cGMP (Refs. 9 and 10 and references therein). Hem-AT-Bs and Hem-AT-Hs are oxygen sensors in which the hemes are thought to mediate signal transduction for methylation of the chemotaxis proteins (11, 12).The Fix proteins, FixL and FixJ, of Rhizobium meliloti are well characterized as biological oxygen sensors and regulate the expression of the nitrogen fixation genes of a plant symbiotic bacterium, Sino...

Binding of Oxygen and Carbon Monoxide to a Heme-regulated Phosphodiesterase from Escherichia coli

“…The phosphodiesterase (PDE) 1 from Escherichia coli, Ec DOS, is composed of an N-terminal heme-bound PAS domain and a C-terminal PDE catalytic domain (1). The basic physicochemical characteristics and function of this enzyme have been partially elucidated by our group and that of Kitagawa et al (1,2).…”

“…The basic physicochemical characteristics and function of this enzyme have been partially elucidated by our group and that of Kitagawa et al (1,2). PDE activity is dependent on the redox state of Ec DOS in that the enzyme is active only when the heme is in the Fe(II) state.…”

“…In these enzymes, association or dissociation of the exogenous axial ligand (O 2 , CO, or NO) from the heme iron leads to protein conformational changes, which in turn transmit signals to other domains to regulate catalysis or binding to DNA. The Ec DOS signal transducing mechanism appears to be unique, since changes in the redox state of the PAS domain, rather than iron coordination chemistry, are responsible for signal transduction (1). However, in other words, signal transduction triggered by ligand binding (CO and NO) is common to Ec DOS and FixL, based on the finding that CO or NO binding abolishes catalysis by Ec DOS (1).…”

“…The Ec DOS signal transducing mechanism appears to be unique, since changes in the redox state of the PAS domain, rather than iron coordination chemistry, are responsible for signal transduction (1). However, in other words, signal transduction triggered by ligand binding (CO and NO) is common to Ec DOS and FixL, based on the finding that CO or NO binding abolishes catalysis by Ec DOS (1).…”

“…: 81-22-217-5604 and 5605; Fax: 81-22-217-5604, 5605, and 5390; E-mail: shimizu@ tagen.tohoku.ac.jp. 1 The abbreviations used are: PDE, phosphodiesterase; Ec DOS, fulllength heme-bound phosphodiesterase from E. coli or a redox sensor from E. coli; PAS, Drosophila period clock protein (PER), vertebrate aryl hydrocarbon receptor nuclear translocator (ARNT), and Drosophila single-minded protein (SIM); FixL, oxygen sensor heme protein from Rhizobium meliloti; CooA, CO sensing protein from Rhodospirillum rubrum; sGC, soluble guanylate cyclase; HemAT, oxygen sensor heme proteins from Bacillus subtilis (HemAT-Bs) and Halobacterium salinarium (HemAT-Hs); AxPDEA1, phosphodiesterase A1 protein from Acetobactoer xylinum; NOR-1, (Ϯ)-(E)-4-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexenamide; FT IR, Fourier transform infrared spectroscopy; Hb, hemoglobin. mutants of Ec DOS as well as infrared spectra of their Fe(II)-CO complexes.…”